Air pulse quantification of supraglottic and pharyngeal sensation: a new technique.

There are no published studies evaluating the sensory capacity of the region innervated by the superior laryngeal nerve. A normal sensory capacity is important in this area, since hypesthesia or anesthesia of the pharynx and supraglottic larynx may result in dysphagia and aspiration. This often occurs after stroke or after ablative surgery of the pharynx and larynx. Evaluating the efficacy of restorative procedures for supraglottic and pharyngeal sensation is dependent on defining and quantifying the sensory deficit. We have developed a new, noninvasive method to measure sensation in the pharynx and supraglottic larynx. A puff of air--of precisely controlled duration and pressure--was delivered via a flexible telescope to the anterior wall of the pyriform sinus. Surface sensibility was determined according to the psychophysical method of limits by varying air pressure while holding puff duration constant. We conducted 204 trials in 20 healthy adults. The average sensory discrimination threshold was 2.09 +/- 0.15 mm Hg. An intraclass correlation revealed excellent consistency (R = .80). There was no statistically significant difference between the right and left sides. Brief air pulse stimulation is an easy, relatively safe, and reliable method of determining supraglottic and pharyngeal sensory discrimination thresholds.

Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role.

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol-induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK.

Cell swelling increases intracellular free [Ca] in cultured toad bladder cells.

We examined the effect of cell swelling on intracellular free calcium concentration ([Ca]i) in cultured toad bladder cells (TB-M) grown as a polarized monolayer on collagen-coated filters. [Ca]i was measured by use of fura-2, fluorescence microscopy, and simple video imaging. In preliminary experiments we determined that reducing ionic strength by 15% had no effect on the Kd of fura-2, indicating that the dye could be used to examine the effects of cell swelling on [Ca]i. Reducing the osmolality of the serosal bathing medium by 15% caused [Ca]i to increase within 10 s from 97 +/- 9 to 354 +/- 88 nM (n = 5). By 2 min [Ca]i had declined to 163 +/- 22 nM. The increase in [Ca]i was not caused by a fall in Na concentration ([Na]) because isosmotic reduction of serosal [Na] did not result in an increase in [Ca]i. The swelling-induced increase in [Ca]i could be abolished by lowering serosal [Ca] to 200 nM and by addition of lanthanum. The calcium-channel blockers nitrendipine and verapamil also inhibited the swelling-induced increase in [Ca]i, although to different degrees. These experiments demonstrate that swelling of cultured toad bladder cells results in a significant increase in [Ca]i by enhancing the rate of calcium entry across the basolateral membrane, possibly through a calcium channel.